1. Field of the Invention
The present invention relates to the identification of new muteins of fibroblast growth factor 21 that have improved pharmaceutical properties.
2. Description of the Related Art
Fibroblast growth factors are large polypeptides widely expressed in developing and adult tissues (Baird et al., Cancer Cells, 3:239-243, 1991) and play crucial roles in multiple physiological functions including angiogenesis, mitogenesis, pattern formation, cellular differentiation, metabolic regulation and repair of tissue injury (McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 59:135-176, 1998). According to the published literature, the FGF family now consists of at least twenty-three members, FGF-1 to FGF-23 (Reuss et al., Cell Tissue Res. 313:139-157 (2003).
Fibroblast growth factor 21 (FGF-21) has been reported to be preferentially expressed in the liver (Nishimura et al., Biochimica et Biophysica Acta, 1492:203-206, (2000); WO01/36640; and WO01/18172) and described as a treatment for ischemic vascular disease, wound healing, and diseases associated with loss of pulmonary, bronchia or alveolar cell function and numerous other disorders. More recently, FGF-21 has been shown to stimulate glucose-uptake in mouse 3T3-L1 adipocytes in the presence and absence of insulin, and to decrease fed and fasting blood glucose, triglycerides, and glucagon levels in ob/ob and db/db mice and 8 week old ZDF rats in a dose-dependant manner, thus, providing the basis for the use of FGF-21 as a therapy for treating diabetes and obesity (WO03/011213). In addition, FGF-21 has been shown to be effective in reducing the mortality and morbidity of critically ill patients (WO03/059270).
A significant challenge in the development of protein pharmaceuticals, such as FGF-21, is to cope with their physical and chemical instabilities. The compositional variety and characteristics of proteins define specific behaviors such as folding, conformational stability, and unfolding/denaturation. Such characteristics must be addressed to stabilize proteins when developing pharmaceutical formulation conditions utilizing aqueous protein solutions (Wang, W., Int. J. of Pharmaceutics, 18, (1999).
Specifically, in pharmaceutical protein development, anti-microbial preservative agents such as phenol, m-cresol, methylparaben, resorcinol, and benzyl alcohol are necessary in parenteral pharmaceutical formulations that are intended to be a sterile, multi-use formulation. Unfortunately, these compounds often adversely affect the stability of the protein product, triggering association and aggregation, in particular (Maa et al., Int. J. of Pharmaceutics 140:155-168 (1996); Lam et al., Pharm. Res. 14(6):725-729 (1997)).
FGF-21 will likely be utilized as a multi-use, sterile pharmaceutical formulation. However, it has been determined that preservatives, i.e. m-cresol, have an adverse affect on its stability under these conditions. Clearly, there is a need to develop a stable aqueous protein formulation for the therapeutic protein FGF-21. The present invention overcomes the significant hurdles of physical instabilities with the invention of muteins of FGF-21 that are more stable than wild-type FGF-21 under pharmaceutical formulation conditions. Thus, the muteins of FGF-21 of the present invention provide stable pharmacological protein formulations that are useful for the treatment of type 2 diabetes, obesity, metabolic syndrome, and in reducing the mortality and morbidity of critically ill patients.